With the development of genetic manipulation, studies on the mass production of useful proteins by the use of bacteria and various animals and plants are frequently being conducted. Up to now, E. coli is a host cell mostly frequently used for the mass production of various useful proteins, studies on which are also most frequently conducted (Choi et al., Chem. Eng. Sci., 66: 876, 2006; Lee, Trends Biotechnol., 14:98, 1996). However, if useful proteins to be produced are produced in the cytoplasm of E. coli, there are many problems. First, in order for proteins produced in the cytoplasm to be isolated and purified with high purity, considerably complex and very expensive isolation and purification procedures should be carried out. And, the proteins are liable to be exposed to various proteases present in the cytoplasm, which leads to a reduction in yield. Also, in most cases, proteins overexpressed in the cytoplasm are not completely folded and form inactive inclusion bodies. Because these inclusion proteins have no activity as proteins, complex and expensive denaturation and refolding procedures are required to obtain biologically active water-soluble proteins from the inclusion proteins (Choi et al., Chem. Eng. Sci., 66: 876, 2006).
As one of methods to solve such problems, there is a method in which proteins produced in the cytoplasm are secreted into the periplasm or cell culture broth. The extracellular production of a target protein in E. coli has various advantages. First, the production of the target protein in E. coli cells can fundamentally prevent intracellular proteolysis, so that the stable production of the desired protein can be expected. Second, by the secretion procedure, the correct folding of proteins is possible so as to produce proteins with activity, and the formation of inclusion bodies by incorrect folding can be prevented. Third, by the secretion process, N-terminal secretion signal sequences are removed so that it is possible to maintain the same amino acid sequences as naturally occurring sequences having no N-terminal methionine (Met) residues which are unavoidably linked in intracellular production. Finally, a purification procedure is easily performed. Since there is little or no protein which is naturally secreted from E. coli into culture broth, the secretory production of the target protein into cell culture broth allows the target protein to be maintained at high purity, so that the pure isolation of the target protein from the cell culture broth becomes very easy. Although the extracellular production of the target protein in E. coli has similar advantages to general secretory production by secretion into the E. coli periplasm, the above-described advantages of the extracellular production can be much more effective in terms of industrial production. Because the extracellular secretory production is a method of completely secreting the target protein to the outside of cells, not secreting the target protein into the limited periplasm, it significantly reduces the load in cells resulting from the overexpression of the protein to allow the mass production of proteins by high cell concentration culture and continuous culture. Although both methods allow proteins with high purity to be produced in a purification procedure, the secretory production of the target protein into the periplasm requires a much more complex procedure than the extracellular secretory production, which is not easy to use in industrial applications (Choi and Lee, Appl. Microbiol. Biotechnol., 64:625, 2004). As described above, the advantages of the extracellular secretion are very excellent and diverse and thus various studies on the periplasmic or extracellular secretion of target proteins in E. coli have been conducted.
Methods of secreting useful proteins synthesized from E. coli into the periplasm or cell culture broth have various advantages in terms of the production of recombinant proteins. First, because the periplasm or culture broth contains a significantly smaller amount of proteins than the cytoplasm does, it is very easy to isolate and purify the desired useful protein with high purity. And, because the useful proteins are separated from the cytoplasm where most of proteases are, intracellular proteolysis can be previously prevented, thus giving good results in terms of yield. Also, because the periplasm is a more oxidized environment than the cytoplasm, disulfide binding is more easily made, so that correct folding of the resulting protein is made, which leads to a remarkable reduction in the formation of inclusion proteins (Choi and Lee, Appl. Microbiol. Biotechnol., 64:625, 2004).
To secrete foreign proteins which are not secreted in E. coli, the previously known secretion signal sequences (e.g., OmpA, OmpF, PhoA, SpA, etc.) were linked to the N-terminal end of foreign proteins or linked after some modification (Abrahmsen et al., EMBO J., 4:3901, 1985; Choi and Lee, Appl. Microbiol. Biotechnol. 64:625, 2004; Jobling et al., Plasmid, 38:158, 1997; Klein et al., Protein Eng., 5:511, 1992; Utsumi et al., Gene, 71:349, 1988).
However, such signal sequences or proteins show a great difference in terms of secretion efficiency and have a number of examples where no secretion is occurred. This is because the correlation between signal sequences and target proteins is not yet clearly established and thus studies on the investigation of new signal sequences capable of efficiently secreting target proteins are still being conducted worldwide. Furthermore, although the isolation and purification of the target protein from the periplasm are easier than the isolation and purification from the cytoplasm, the extracellular secretion of the target protein can make the isolation and purification of the target protein easier.
Methods for the extracellular secretion of target proteins in E. coli, which have been performed till now, can be broadly divided into the following four categories:    (1) The first method is based on a fusion protein combining a signal peptide and the target protein and mainly uses the signal peptide to secrete target proteins from E. coli to the periplasm. Toksoy et al. reported the extracellular production of a TaqI protein by fusion with a maltose binding protein (MBP) (Toksoy et al., Biotechnol Techniq., 3:803, 999). In this case, an E. coli XL1 strain was used, and after the induction of expression with 1 mM IPTG, about 270×103 units/IL culture broth of the TaqI protein was produced in cell culture broth. Lo et al. reported that β-1,4-endoglucanase derived from Bacillus subtilis was expressed in E. coli, as a result, the extracellular secretion of the protein occurred (Lo et al., Appl. Environ. Microbiol., 54:2287, 1988). Nagahari et al. reported the extracellular secretion of β-endorphin by fusion with the secretion signal sequence and eight N-terminal amino acids of the OmpF protein (Nagahari et al., EMBO J., 4:3589, 1985). In this case, E. coli N99, RRI, and MC4100 and their mutants MH1461 (envZ) and MH1160 (ompR) were used as host cells, and among them, the RR1 strain showed the highest secretion efficiency, in which the degradation of β-endorphin occurred by the action of extracellular protease after secretion. The most stable secretion efficiency was shown in the N99 strain. On the other hand, Yamamoto et al. attempted the secretory production of Harvey murine sarcoma virus-derived p21 protein by fusion with the OmpF secretion signal sequence as mentioned above. However, the p21 protein was not secreted to the outside of cells, but accumulated in the cytoplasm in the form of insoluble inclusion bodies (Yamamoto et al., Appl. Microbiol. Biotechnol., 35:615, 1991).    (2) The second method uses the co-production of a target protein and a protein involved in the secretion of the target protein in E. coli. Baneyx et al. reported the co-production of a TolAIII protein (E. coli transmembrane protein) in the secretory production of an OmpA-TEM-β-lactamase fusion protein (Baneyx and Eugene, Protein Expr Purif., 14:13, 1998). For the secretory production of the OmpA-TEM-β-lactamase fusion protein, a lpp-lac promoter requiring IPTG derivatives was used, and for the expression of the TolAIII protein, a T7lac promoter in need of the IPTG derivatives was used. The expression of the protein in E. coli BL21 (DE3) was induced with 1 mM IPTG. As a result, the co-expression with TolAIII showed a 3.5 times increase in the activity of β-lactamase as compared to the single expression of the protein. Robbens et al. reported the co-expression of a kil gene in the production of interleukin-2 (IL-2) (Robbens et al., Protein Expr Purif., 6:481, 1995). In this case, after the IL-2 gene was fused with an OmpA secretion signal sequence, its expression was induced with a tac promoter in need of the IPTG derivatives, and the co-expression of the kil gene used a PL promoter requiring heat shock at 42° C. It was seen that the IL-2 protein produced in the E. coli periplasm before the induction of expression of the kil gene by heat shock was mostly secreted into cell culture broth through the outer membrane after the induction of expression of the kil gene. van der Wal et al. used bacteriocin release protein (BRP) which is an E. coli lipoprotein, for the extracellular secretion of β-lactamase (van der Wal et al., Appl. Environ. Microbiol., 64:392, 1998). van der Wal et al. obtained modified BRP genes by the random mutagenesis of the existing BRP gene, and used these modified BRP genes to develop systems which can secrete and produce to the outside of cells in a more stable manner than the existing BRP gene. Aristidou et al. reported that the addition of glycine into medium was effective for increasing secretion efficiency in extracellular secretion with the use of BRP (Aristidou et al., Biotechnol. Lett., 15:331, 1993). As target proteins, α-amylase and β-lactamase were used, and the expression of BRP was induced by a lpp/lac promoter. The α-amylase showed about 10 times higher activity with the addition of 1% glycine into the medium than with the addition of 0.1% glycine. The β-lactamase showed about 2.5 times increase in its activity with the addition of 1.0% glycine.    (3) The third method uses an E. coli strain with no outer membrane. This mutant, so called a “L-form”, is in a state where the E. coli outer membrane was removed so that the cells were formed, having the inner membrane without the periplasm. Namely, the use of a method for secreting proteins to the periplasm, which has been widely used in the prior art, allows the extracellular production of proteins, because once the proteins are passed through the inner cell membrane, they are exposed to cell culture broth without passing through the periplasm. Kujau et al. used a RV308 strain which is L-form E. coli strain, for the extracellular secretion of a miniantibody (miniAb) (Kujau et al., Appl. Microbiol. Biotechnol., 49:51, 1998). The miniAb gene was fused with the OmpA secretion signal sequence, after which its expression was induced by the lac promoter. Culturing at a low temperature of 26° C. showed a higher cell concentration and protein production than culturing at 37° C.    (4) The fourth method is to perform extracellular secretion by the fusion of outer membrane protein F (OmpF) with a target protein. In this method, target protein β-endorphin fused with the C-terminal end of the outer membrane protein F was cultured at high cell concentration so as to be secreted to the outside of cells (Korean patent registration No. 10-0447530; Jeong and Lee, Appl. Environ. Microbiol., 68:4979, 2002). However, this method has problems in that high cell concentration culture must be carried out for the extracellular secretion of the target protein, and a step of isolating and purifying the target protein by digestion with various proteases is required because the produced target protein is fused with the OmpF protein.
As described above, although various methods for the extracellular production of a target protein in E. coli have been developed, various problems still remain. The above-described methods allow the secretory production of a target protein. However, because most of these methods involve partial lysis of E. coli, significant amounts of intracellular proteins of E. coli are contained in cell culture broth to reduce the purity of the target protein, thus making it difficult to achieve an easy purification procedure which is the greatest advantage of extracellular secretory production. Also, because these methods involve the lysis of E. coli, they have a shortcoming in that high cell concentration culture is difficult so that it also becomes difficult to achive the mass production of the desired protein. Particularly in L-form E. coli, only low cell concentration culture is possible because of the structural problem of E. coli strains.